Abstract
Purpose: :
Administration of AAV vector into small rodent eyes, because of the large injection volumes relative to the size of the eye, may damage and/or evoke stress responses not seen in larger eyes. In order to test the therapeutic usefulness of lens epithelium–derived growth factor (LEDGF) in murine models of retinal degeneration, we employed two alternative, systemic routes of vector administration, either intra–muscular or intra–peritoneal.
Methods: :
The vector included a LEDGF–GFP fusion transgene under CMV promoter regulation and was packaged into serotype 5 AAV particles. 10exp12 AAV5.CMV.EGFP–LEDGF particles were injected intra–muscularly (IM) into the each caudal muscle of the pelvic limb of rd12 mice at PN30, and 4X10exp12 particles were injected intra–peritoneally (IP) into rd12 or rd10 mice at PN3–5 or PN30. Accumulation of LEDGF–GFP protein in retinas was evaluated by immunocytochemistry and western–blot hybridization using both GFP and LEDGF antibodies. Liver, kidney, spleen, lung and heart were also analyzed by western–blotting and RT PCR to more completely determine the distribution of LEDGF–GFP expression/production. Rd10 mice injected IP (n=3) and the control counterparts of the same age were tested by scotopic ERG.
Results: :
Both IP and IM AAV–vectored LEDGF–GFP transgene administration resulted in protein accumulation in retinas of either Rd12 or Rd10 mice at all ages tested. GFP positive signals were detected in retinal blood vessels 1week post–injection. GFP reactivity was sporadically seen in cells of the retinal proper at the later times. LEDGF–GFP was also seen in livers when vector was delivered IM and in liver and kidney after IP administration. Preliminary ERG results provide evidence for functional rescue in rd10 mice treated at PN30, but as yet no evidence of the morphological improvement has been noted.
Conclusions: :
Both, IM and IP AAV–vectored transgene administration are effective routes for transferring LEDGF to the murine retina. The LEDGF–GFP fusion protein was found in retinal blood vessels by1week post–injection with an increase of sporadically positive retinal cells at later times. Given the history of liver expression of systemic AAV–delivered transgenes and our detection of LEDGF transgene product there, the liver is the most likely target for the AAV–vectored protein production following an IP route of administration.
Keywords: gene transfer/gene therapy • immunohistochemistry • retina